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Glutamate Decarboxylase: Loss of N-terminal Segment Does Not Affect Homodimerization and Determination of the Oxidation State of Cysteine Residues

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Glutamate decarboxylase (GAD) produces GABA, the main inhibitory neurotransmitter in adult mammalian brain. The physical characteristics of GAD were studied using mass spectrometry and partial protein digests. The N-termini of the two main isoforms, GAD65 and GAD67, were processed by removal of the initial methionine residues and acetylation of the penultimate alanines. Native recombinant GAD65 and GAD67 exist as homodimers that can be dissociated with non-reducing methods, indicating that homodimerization does not involve intermolecular disulfide bonds. Truncation of the N-terminal segment with trypsin digestion did not affect homodimerization but increased activity by decreasing the Km of GAD67 and increasing the Vmax of both isoforms. Of the 15 cysteines in GAD65, the six found in the N-terminal segment can form disulfide bonds and of the 13 cysteines in GAD67, cysteines 32 and 38 can form a disulfide bond. The in vitro formation of disulfide bonds in the N-termini, and the removal of the termini with relatively low amounts of trypsin, indicate that the N-terminal segments of GAD65 and GAD67 are exposed and flexible. The formation of a disulfide bridge between cysteines 30 and 45 of GAD65 suggests that alteration of normal redox conditions could affect GAD targeting.

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References

  1. S. Baekkeskov H. J. Aanstoot S. Christgau A. Reetz M. Solimena M. Cascalho F. Folli H. Richter-Olesen P. Camilli ParticleDe P. D. Camilli (1990) ArticleTitleIdentification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase Nature 347 151–156 Occurrence Handle10.1038/347151a0 Occurrence Handle1697648

    Article  PubMed  Google Scholar 

  2. J. W. Yoon R. S. Sherwin H. Kwon H. S. Jun (2000) ArticleTitleHas GAD a central role in type 1 diabetes? J. Autoimmun. 15 273–278 Occurrence Handle10.1006/jaut.2000.0442 Occurrence Handle11040067

    Article  PubMed  Google Scholar 

  3. D. -F. Bu M. G. Erlander B. C. Hitz N. J. K. Tillakaratne D. L. Kaufman C. B. Wagner-McPherson G. A. Evans A. J. Tobin (1992) ArticleTitleTwo human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene Proc. Natl. Acad. Sci. USA 89 2115–2119 Occurrence Handle1549570

    PubMed  Google Scholar 

  4. M. G. Erlander N. J. K. Tillakaratne S. Feldblum N. Patel A. J. Tobin (1991) ArticleTitleTwo genes encode distinct glutamate decarboxylases Neuron 7 91–100 Occurrence Handle10.1016/0896-6273(91)90077-D Occurrence Handle2069816

    Article  PubMed  Google Scholar 

  5. D. L. Kaufman C. R. Houser A. J. Tobin (1991) ArticleTitleTwo forms of the gamma-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions J. Neurochem. 56 720–723 Occurrence Handle1988566

    PubMed  Google Scholar 

  6. M. Esclapez N. J. K. Tillakaratne D. L. Kaufman A. J. Tobin C. R. Houser (1994) ArticleTitleComparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms J. Neurosci. 14 1834–1855 Occurrence Handle8126575

    PubMed  Google Scholar 

  7. M. Namchuk L. Lindsay C. W. Turck J. Kanaani S. Baekkeskov (1997) ArticleTitlePhosphorylation of serine residues 3, 6, 10, and 13 distinguishes membrane anchored from soluble glutamic acid decarboxylase 65 and is restricted to glutamic acid decarboxylase 65α J. Biol. Chem. 272 1548–1557 Occurrence Handle10.1074/jbc.272.3.1548 Occurrence Handle8999827

    Article  PubMed  Google Scholar 

  8. S. Christgau H. J. Aanstoot H. Schierbeck K. Begley S. Tullin K. Hejnaes S. Baekkeskov (1992) ArticleTitleMembrane anchoring of the autoantigen GAD65 to microvesicles in pancreatic beta-cells by palmitoylation in the NH2-terminal domain J. Cell Biol. 118 309–320 Occurrence Handle10.1083/jcb.118.2.309 Occurrence Handle1321158

    Article  PubMed  Google Scholar 

  9. D. L. Martin K. E. Barke (1998) ArticleTitleAre GAD65 and GAD67 associated with specific pools of GABA in brain? Perspect. Dev. Neurobiol. 5 119–129 Occurrence Handle9777630

    PubMed  Google Scholar 

  10. J. Kanaani Ael el Husseini A. Aguilera-Moreno J. M. Diacovo D. S. Bredt S. Baekkeskov (2002) ArticleTitleA combination of three distinct trafficking signals mediates axonal targeting and presynaptic clustering of GAD65 J. Cell Biol. 158 1229–1238 Occurrence Handle10.1083/jcb.200205053 Occurrence Handle12356867

    Article  PubMed  Google Scholar 

  11. J. Kanaani D. Lissin S. F. Kash S. Baekkeskov (1999) ArticleTitleThe hydrophilic isoform of glutamate decarboxylase, GAD67, is targeted to membranes and nerve terminals independent of dimerization with the hydrophobic membrane-anchored isoform, GAD65 J. Biol. Chem. 274 37200–37209 Occurrence Handle10.1074/jbc.274.52.37200 Occurrence Handle10601283

    Article  PubMed  Google Scholar 

  12. R. Dirkx SuffixJr. A. Thomas L. Li Å. Lernmark R. S. Sherwin P. Camilli ParticleDe M. Solimena (1995) ArticleTitleTargeting of the 67-kDa isoform of glutamic acid decarboxylase to intracellular organelles is mediated by its interaction with the NH2-terminal region of the 65-kDa isoform of glutamic acid decarboxylase J. Biol. Chem. 270 2241–2246 Occurrence Handle10.1074/jbc.270.5.2241 Occurrence Handle7836456

    Article  PubMed  Google Scholar 

  13. F. W. Alexander E. Sandmeier P. K. Mehta P. Christen (1994) ArticleTitleEvolutionary relationships among pyridoxal-5′-phosphate-dependent enzymes. Regio-specific alpha, beta and gamma families Eur. J. Biochem. 219 953–960 Occurrence Handle10.1111/j.1432-1033.1994.tb18577.x Occurrence Handle8112347

    Article  PubMed  Google Scholar 

  14. C. Momany R. Ghosh M. L. Hackert (1995) ArticleTitleStructural motifs for pyridoxal-5′-phosphate binding in decarboxylases: An analysis based on the crystal structure of the Lactobacillus 30a ornithine decarboxylase Protein Sci. 4 849–854 Occurrence Handle7663340

    PubMed  Google Scholar 

  15. K. Qu D. L. Martin C. E. Lawrence (1998) ArticleTitleMotifs and structural fold of the cofactor binding site of human glutamate decarboxylase Protein Sci. 7 1092–1105 Occurrence Handle9605314

    PubMed  Google Scholar 

  16. S. N. Sheikh D. L. Martin (1996) ArticleTitleHeteromers of glutamate decarboxylase isoforms occur in rat cerebellum J. Neurochem. 66 2082–2090 Occurrence Handle8780039

    PubMed  Google Scholar 

  17. D. L. Martin (1986) Brain glutamate decarboxylase A. A. Boulton G. B. Baker P. H. Yu (Eds) Neuromethods, vol. 5, Neurotransmitter Enzymes Humana Press Clifton, NJ 361–388

    Google Scholar 

  18. G. Battaglioli H. Liu D. L. Martin (2003) ArticleTitleKinetic differences between the isoforms of glutamate decarboxylase: implications for the regulation of GABA synthesis J. Neurochem. 86 879–887 Occurrence Handle10.1046/j.1471-4159.2003.01910.x Occurrence Handle12887686

    Article  PubMed  Google Scholar 

  19. S. B. Martin D. L. Martin (1979) ArticleTitleStimulation by phosphate of the activation of glutamate apodecarboxylase by pyridoxyl-5'-phosphate and its implications for the control of GABA synthesis J. Neurochem. 33 1275–1283 Occurrence Handle552405

    PubMed  Google Scholar 

  20. U. K. Laemmli (1970) ArticleTitleCleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227 680–685 Occurrence Handle10.1038/227680a0 Occurrence Handle5432063

    Article  PubMed  Google Scholar 

  21. K. Rimvall S. Sheikh D. L. Martin (1993) ArticleTitleEffects of increased gamma-aminobutyric acid levels on GAD67 protein and mRNA levels in rat cerebral cortex J. Neurochem. 60 714–720 Occurrence Handle8419546

    PubMed  Google Scholar 

  22. S. N. Sheikh S. B. Martin D. L. Martin (1999) ArticleTitleRegional distribution and relative amounts of glutamate decarboxylase isoforms in rat and mouse brain Neurochem. Int. 35 73–80 Occurrence Handle10.1016/S0197-0186(99)00063-7 Occurrence Handle10403432

    Article  PubMed  Google Scholar 

  23. J. Wu J. T. Watson (1997) ArticleTitleA novel methodology for assignment of disulfide bond pairings in proteins Protein Sci. 6 391–398 Occurrence Handle9041641

    PubMed  Google Scholar 

  24. Y. C. Chang D. I. Gottlieb (1988) ArticleTitleCharacterization of the proteins purified with monoclonal antibodies to glutamic acid decarboxylase J. Neurosci. 8 2123–2130 Occurrence Handle3385490

    PubMed  Google Scholar 

  25. J. Wei K. M. Davis H. Wu J. Y. Wu (2004) ArticleTitleProtein phosphorylation of human brain glutamic acid decarboxylase (gad)65 and gad67 and its physiological implications Biochemistry 43 6182–6189 Occurrence Handle10.1021/bi0496992 Occurrence Handle15147202

    Article  PubMed  Google Scholar 

  26. J. Kim M. Namchuk T. Bugawan Q. Fu M. Jaffe Y. Shi H.-J. Aanstoot C. W Turck H. Erlich V. Lennon S. Baekkeskov (1994) ArticleTitleHigher autoantibody levels and recognition of a linear NH2-terminal epitope in the autoantigen GAD65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus J. Exp. Medicine 180 595–606 Occurrence Handle10.1084/jem.180.2.595

    Article  Google Scholar 

  27. L. G. Hom L. E. Volkman (1998) ArticleTitlePreventing proteolytic artifacts in the baculovirus expression system BioTechniques 25 18–20 Occurrence Handle9668966

    PubMed  Google Scholar 

  28. D. L. Martin (1987) ArticleTitleRegulatory properties of brain glutamate decarboxylase Cell. Mol. Neurobiol. 7 237–253 Occurrence Handle10.1007/BF00711302 Occurrence Handle3326683

    Article  PubMed  Google Scholar 

  29. Y. Shi B. Veit S. Bækkeskov (1994) ArticleTitleAmino acid residues 24–31 but not palmitoylation of cysteines 30 and 45 are required for membrane anchoring of glutamic acid decarboxylase, GAD65 J. Cell Biol. 124 927–934 Occurrence Handle10.1083/jcb.124.6.927 Occurrence Handle8132714

    Article  PubMed  Google Scholar 

  30. M. Solimena R. Dirkx SuffixJr. M. Radzynski O. Mundigl P. Camilli ParticleDe (1994) ArticleTitleA signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region J. Cell Biol. 126 331–341 Occurrence Handle10.1083/jcb.126.2.331 Occurrence Handle8034738

    Article  PubMed  Google Scholar 

  31. J. Wei Y. Jin H. Wu D. Sha J. Y. Wu (2003) ArticleTitleIdentification and functional analysis of truncated human glutamic acid decarboxylase 65 J. Biomed. Sci. 10 617–624 Occurrence Handle10.1159/000073527 Occurrence Handle14576464

    Article  PubMed  Google Scholar 

  32. C.-H. Chen S. J. Wu D. L. Martin (1998) ArticleTitleStructural characteristics of brain glutamate decarboxylase in relation to its interaction and activation Arch. Biochem. Biophys. 349 175–182 Occurrence Handle10.1006/abbi.1997.0457 Occurrence Handle9439596

    Article  PubMed  Google Scholar 

  33. K. J. Barnham C. L. Masters A. I. Bush (2004) ArticleTitleNeurodegenerative diseases and oxidative stress Nat. Rev. Drug Discov. 3 205–214 Occurrence Handle10.1038/nrd1330 Occurrence Handle15031734

    Article  PubMed  Google Scholar 

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Correspondence to Gino Battaglioli.

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Battaglioli, G., Liu, H., Hauer, C.R. et al. Glutamate Decarboxylase: Loss of N-terminal Segment Does Not Affect Homodimerization and Determination of the Oxidation State of Cysteine Residues. Neurochem Res 30, 989–1001 (2005). https://doi.org/10.1007/s11064-005-6772-0

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